Gene transfer is a powerful tool for the analysis of factors which control gene expression in mammalian cells. We have transferred the Herpes simplex virus thymidine kinase (TK) gene into mouse LTK cells and have obtained cell lines which physically retain the genes but modulate expression when selection against TK is applied. The efficient selections for and against expression of TK coupled with the use of the cloned viral gene as a nucleic acid probe provide a powerful system with which to correlate changes in DNA and/or chromatin structure with gene expression. We propose to analyze several cell lines, one of which switches from TK+ to Tk- and back at a frequency of 10 to the minus 6, and one which undergoes the same phenotypic changes at a frequency of 10 to the minus 2, for changes in DNA which correlate with and cause changes in gene expression. In a second set of experiments, the normal 5 feet flanking dNA (promoter region) of the TK gene has been replaced with randomly cloned mouse DNA, and several sequences which restore the ability of the gene to function in gene transfer experiments have been identified. We propose to isolate and characterize several such sequences to elucidate features which are important for gene expression. The final set of experiments will determine if a mutant cellular gene can be rescued by recombination with an incoming gene. These experiments may point out ways to perform gene transfer experiments to ask more sophisticated questions than currently possible.